1. Field of the Invention
This invention relates to methods in which x-ray topographic images of protein crystals are made and analyzed to identify conditions for growing protein crystals having greater order and fewer crystal defects that are suitable for use in determining the structure of the protein by x-ray diffractometry (crystallography).
2. Description of Related Art
X-ray topography is a well-established technique for characterizing and studying, by means of x-ray diffraction, the defect microstructure of single-crystals. Historically, the technique has largely been used to detect defects in inorganic crystalline structures (Bowen, 1998 and Tanner, 1976). More recently, x-ray topography has been employed to detect imperfections in protein crystals (for example, see Caylor et al., 1999).
The present invention fulfills a long-felt need for improved methods for producing good-quality protein crystals, by providing methods in which x-ray topographic analysis is systematically applied to identify conditions giving improved crystal growth. Of particular importance, the present invention uses x-ray topographic analysis in conjunction with two powerful, new methods for producing high-quality protein crystalsxe2x80x94the use of dynamically controlled crystallization for crystallizing proteins, and the crystallization of proteins at effective gravity geff that is different from gravity at the earth""s surface.
The former approach involves crystallizing protein using a dynamically controlled crystallization system (DCCS) having a crystallization chamber that contains the protein in a buffered solution separated from the precipitating solution by a dialysis type membrane. A computer-controlled mechanism gradually changes the precipitating solution strength in a very precisely controlled manner, thereby causing the protein to leave its solution, form aggregates, nucleate, and initiate crystal growth.
The latter approach can be accomplished by growing the crystals in space, or in a magnetic field that causes the protein molecules in the crystal to experience an effective gravitational field that is greater or less than the gravitational field at the earth""s surface.
The present invention provides a general method and system for identifying conditions for growing protein crystals having greater order and fewer crystal defects that are suitable for use in determining the structure of the protein by x-ray crystallography (diffractometry). Crystals of a protein are grown under different sets of predetermined conditions, and x-ray topographic images of the protein crystals are generated. The x-ray topographic images reveal defects in the crystals, and permit identification of the set(s) of conditions that produce crystals having the fewest crystal defects. In a preferred embodiment, the protein crystals are grown in a dynamically controlled crystallization system (DCCS). An important condition of crystal growth that can be optimized by the method is the effective gravity, geff, experienced by the growing crystal; for example, when the crystal is grown under microgravity in space, or in a powerful magnetic field that causes the protein molecules in the growing crystal to experience acceleration of an effective gravitational field that is greater or less than the actual gravitational field at the earth""s surface. With the present method, it is possible to identify differences between crystals grown on the earth with the DCCS, and those grown in space under identical conditions. A comparison of x-ray topographs taken from both earth grown and space grown crystals indicates that the space grown crystals are of higher crystallographic perfection. X-ray topography can similarly be used to identify a condition of effective gravity geff within a magnetic field that is less than the gravitational field at the earth""s surface at which a protein crystal having fewer defects can be grown.
The method of the present invention thus comprises identifying conditions at which a protein crystal having the fewest defects can be grown, growing a protein crystal under those conditions, and subjecting such a crystal to x-ray crystallography to solve the structure of the crystallized protein, with the result that the structure so obtained is more accurate than the structure that would be obtained from a crystal having more defects.